Profile Analysis of the Proteome of the Egg of the High Royal Jelly Producing Bees (Apis mellifera L.)

Available online at www.sciencedirect.com

Agricultural Sciences in China

2007, 6(9): 1138-1148

ScienceDirect

September 2007

ProfileAnalysis of the Proteome of the Egg of the High Royal Jelly Producing Bees (Apis mellifera L.) ZHANG L a n 1 . 2 , LI Jim-ke*and WU Li-ming2 1 Department of Bioengineering, Zhengzhou University, Zhengzhou 450001, P.R.China zlnstitute of Apicultural Research, Chinese Academy of Agricultural Sciences, Beijing 100093, P.R.China

Abstract The protein composition of the egg development in the high royal jelly producing bees (Apis mellifera L.) was investigated. This pioneer study was to separate and quantify the proteins in the egg of the high royal jelly producing worker bees (Apis mellijera L.) by using two-dimensional gel electrophoresis along with their three-day development. The results showed that 160, 195, and 176 proteins, with a wide range of molecular weight (17-80 m a ) and relatively narrow scope of PI (4. 00-8.40) could be detected on day 1, day 2, and day 3, respectively, during the developmental process of the egg. Meanwhile 44 protein spots were constantly detected along with the egg development. Among them 36% were in the uptrend along with the egg development, 14% were in the downtrend, and 39% were of the largest expressed volume on day 2. In addition, the specific proteins were expressed on day 1, day 2, and day 3 (89, 77, and 80, respectively). Besides the coexistent and specific proteins, 24 proteins were expressed on day 1 and day 2, but silenced on day 3,49 proteins were expressed on day 2 and day 3, but silenced on day 1, only 3 proteins were expressed on day 1 and day 3, but silenced on day 2. The result indicates that egg development is a sequential and complex gene controlled process, where the eggs of day 2 express the most active proteins. The coexistent proteins suggest that it is conservative and indispensable for this event. These specific proteins suggest that the different developmental stage needs specific proteins to regulate it.

Key words: honeybees, high royal jelly producing, worker bee’s egg, two-dimensional gel electrophoresis, proteome.

INTRODUCTION Royal jelly is not only a natural food benefiting human beings’ health, but also a lucrative hive-product for beekeepers. Royal jelly, bred in the 1980s in China, is mainly produced in China, with an annual collection of about 2000 tons, accounting for 90% of the world’s total output, which is attributed to the high royal jelly producing bees (Apis mellifera L.). Indeed, it is the rare genic resource in China and even in the world. Honeybees (Apis mellifera) are eusocial insects

belonging to Hymenoptera. They work together in a highly structured social order. Each bee belongs to one of the three specialized groups called castes. The different castes are: queen bees, drones, and worker bees. All the castes are holometabolicinsects with the same four stages, egg, larva, pupa, and adult, during their development. The queen lays the egg and stands at the bottom of the cell. Before hatching into the larval stage, the egg period is approximately 72 h (Graham 1992). To study the egg development of the high royal jelly producing bees, it will be conducive to find out the

This paper is translated from its Chinese version in Scientiu Agriculturu Sinica ZHANG Lan, MSc, E-mail: [email protected];Correspondence LI Jim-ke. Tel: +86-10-62591449, E-mail: [email protected]

02007, CAAS.All lightsmsenred.Publishedby Elsevterltd.

Profile Analysis of the Proteome of the Egg of the High Royal Jelly Producing Bees (Apis mellifera L.)

molecular mechanism concerning the high royal jelly production and lay a foundation for molecular breeding. As the high royal jelly producing bees (Apis mellifera L.) were bred from the local Italian bees (Apis mellifera L.) in the 1980s, in China (Chen et al. 2005; Li and Wang 2005), and has become the best royal jelly producer in the world and gained more and more attention. In the past decades, the high royal jelly producing bees (Apis mellifera L.) were studied extensively, ranging from phenotypic investigation to molecular biology assay. During the period from the late 1980s to the late 1990s, a wide spectrum of field investigations showed that the production of the high royal jelly producing bees (Apis mellifera L.) was significantly higher than that of their unselected counterpart, Italian bees (Apis mellifera L.) (Chen and Han 1992; Shen and Xiao 1993; Chen and Lin 1995; Xu et al. 2000, 2001; Liu et al. 2001). From the late 1990s to the early 2000s, the bursa number, weight, and length of the hypopharyngeal gland were reported to be the morphological genetic markers of the high royal jelly producing worker bees (Apis mellifera L.) (Su 2000; Su and Chen 2003). At the same time, some DNA markers of the high royal jelly producing bees (Apis melEifera L.) were also reported by using molecular biological technologies (Zhang et al. 2001a, b, c; Jiang et al. 2002; Wang et al. 2002; Dai et al. 2003; Jin et al. 2003, 2004). Meanwhile, the genetic studies show that the royal jelly producing traits were dominated by more than 70% of the genetic component, by employing the genotypic model (Li et al. 2003a, b). Subsequently, the DNA microsatellite analysis had indicated that seven alleles were greatly related to the high royal jelly production (Li and Wang 2005). However, no studies have been done on the development process, from zygotes (eggs) to brood emergence, of the high royal jelly producing bees (Apis mellifera L,), although a few reports are available on the egg of the honeybees (Apis melliferu), which are about expression patterns of deformed proteins during embryogenesis of honeybees (Apis mellifera L.) (Fleig et al. 1992), the behavior of sperm from egg penetration till the creation of the zygote, the development of the maternal pronucleus, the first two cleavage divisions (Ronglin and Omholt 1999), and the ultrastructure surface of the queen-laid diploid and

1139

haploid eggs, and that of worker-laid eggs (Tamar et al. 2003). Therefore, the purpose of this study is to add some data on how protein is expressed during the egg developmental process of the high royal jelly producing worker bees (Apis mellifera L.), by using the proteomics approach, which will be conducive to find out the character of protein expression and regulation along with the egg development of the high royal jelly producing bees (Apis mellifera L.), and lay a foundation for clarifying the molecular mechanism of high royal jelly production.

MATERIALS AND METHODS Chemicals Immobilized pH gradients (IPG)strip, two-dimensional gel electrophoresis (2-DE) marker and Bio-lyte (pH 310) were purchased from Bio-Rad Laboratories Inc., USA. Tris-base, ammonium persulfate (AP), sodium dodecyl sulfate (SDS), and glycine were bought from Sigma, USA. Acrylamide,N,N-methylenebisacrylamide, bromphenol blue, coomassie brilliant blue (CBB) R-250, coomassie brilliant blue (CBB) G-250, thiourea, 3-[(3cholamidopropy1)-dimethylammonio]-1-propane sulfonate (CHAPS), bovine serum albumin (BSA), agarose and urea were purchased from Amresco., USA. 2,3-dihydroxybutane- 1,4-dithiol (DTT), and iodoacetoamide were purchased from Merck., Germany. All other chemical reagents were from Beijing Chemical Reagents Inc., China.

Honeybeeeggs Specific age eggs of the worker bees of the high royal jelly producing bees (Apis mellifera L.), on day 1, day 2, and day 3, were randomly collected from the queencontrolled frame in the Experimental Apiary of Institute of Apicultural Research, Chinese Academy of Agricultural Sciences in Beijing, China. To guarantee the exact age, the eggs could be sampled. The queen was confined to a confinement chamber (the queencontrolled frame) where only one empty frame could be put in. Then the queen was allowed to lay her eggs

02007,CAAS. All tights reserved. Publishedby Elsevier Ltd.

ZHANG Lan e l al.

1140

into the cells on the frame for 24 h. Subsequently, the queen was removed from the chamber after 24 h and the worker bees’ eggs of day 1 were collected with a plastic transfer tool and put into the frame confinement, to which the queen was forbidden to access. By the same method, the worker bees’ eggs of day 2 and day 3 were collected before 48 and 72 h, respectively. A total of 150 eggs of the worker bees were sampled for each age.

extraction of the worker bees’ eggs, was stored at -70°C for further use.

Protein extraction

Two-dimensional gel electrophoresis (2-DE)

Protein extraction was performed according to the method of Zhong e t a l . (2005), with some buffer) improvements. The eggs (1 mg eggs, 10 pL-’ were mixed in a phosphate buffer (PB) pH 7.6, containing 32.5 mM K,HPO,, 2.6 mM KH,PO,, and 400 mM NaC1. The mixture was homogenized for 20 rnin in the ice and sonicated for 2 min, then centrifuged at 12 000 g and 4°C for 10 min, and further centrifuged at 15000 g and 4°C for 10 min. The supernatant was removed to another tube for further use. The pellets (1 mg eggs, 2 pL-’buffer) were mixed in the aforementioned PB pH 7.6, and then centrifuged at 15000 g and 4°C for 10 min. The supernatant was removed and mixed into the above tube, containing the supernatant as PB-dissolubleprotein extraction, whereas, the pellets (I mg eggs, 10 pL-’buffer) and PB-indissolubleproteins were mixed in a lysis buffer composed of 8 M urea, 2 M thiourea, 4% CHAPS, 20 mM Tris-base, 30 mM DTT, and 2% Bio-lyte (pH 3-10), and then the mixture was homogenized for 10 rnin in ice, sonicated for 2 min, and then centrifuged at 15 000 g and 4°C for 10 min. The supernatant was removed and mixed into the above-mentioned tube containing PB-dissoluble protein extraction, and the debris was discarded. Trichloroacetic (TCA) was added to the collected supernatants to a final concentration of lo%, and then the mixture was kept in ice for 10 min for precipitating proteins and desalting. Subsequently, the mixture was centrifuged twice at 15000 g and 4°C for 10 min. The supernatant was discarded and the pellets (1 mg eggs, 5 pL-’buffer) were resolved in the foregoing lysis buffer, and then the mixture was homogenized for 5 rnin in ice and sonicated for 2 min. Subsequently it was adjusted to pH 7.0 with 2 M NaOH. The mixture, the protein

Thirty micro liters of the protein extraction of the worker bees’ eggs was suspended in 120 pL of the rehydration buffer (8 M urea, 4% CHAPS, 0.001% bromophenol blue, 65 mM DTT, 0.2% Bio-lyte pH 310). A mixture of 125-150 pL (each sample containing 77.4 pg of protein) was loaded on a 7 cm immobilized pH gradient (IPG) strip (pH 3-10 L) and isoelectric focusing (IEF) was performed at 18°C on a Protean IEF cell system (Bio-RadHercules, CA, USA) according to the following program: 14 h at 50 V, 30 rnin of a linear gradient at 250 V twice, 30 rnin at 500 V, 3 h of a linear gradient at 4000 V, and 20000 V h-’ at 4000 V. Then the strip was equilibrated in equilibration buffer 1, containing 6 M urea, 0.375 M Tris-HC1 (pH 8.8), 20% glycerol, 2% SDS, 2% DTT for 15 min, and then continued in the equilibration buffer 2, containing 6 M urea, 0.375 M Tris-HC1 (pH 8.8), 20% glycerol, 2% SDS, 2.5% iodoacetoamide for 15 min. After equilibration, the strip was transferred to SDSPAGE gel, 12% T separating gel (0.75 mm thick). Meanwhile 5 pL of the 2-DE marker was loaded onto a piece of filter paper, and then it was transferred adjacently to the acid tip of the strip when the filter paper was nearly dry. The second dimension electrophoresis, SDS-PAGE, was performed on a mini protean 3 cell (Bio-Rad Hercules, CA, USA) according to the following program: Ten rnin at 80 V, and 200 V until bromophenol blue was running out of the gel at room temperature. Then the gel was stained with CBB R-250.

Protein determination Protein concentration was determined according to the method developed by Bradford (1976), using BSA as the standard. The absorption was measured at 595 nm (Beckman, spectrophotometer DUSOO).

Image analysis The gel was scanned using a transparency mode

02007,CAAS. All rightsreserved. Publishedby Elsevier Ltd.

Profile Analvsis of the Proteome of the Eee. of the Hieh Roval Jellv Producing Bees (Aois mellifera L.)

scanner, connected to the PC system, at 32-bit redgreen-blue colors and 500 dpi resolution for documentation. The image was analyzed using PDQuest 7.3.0 (Bio-Rad Hercules, CA, USA).

RESULTS Profile of 2-DE of the eggs of the high royal jelly producing worker bees (Apis rnellifera L.) The results were reproducible because they have been duplicated for five times. The three protein patterns

1141

corresponding to the eggs of day 1, day 2, and day 3 are shown in Fig. 1. All the three gels had been analyzed with the same parameters, 16.93 of sensitivity, 5 of size scale (PDQuest 7.3.0). With the method and condition of the experiment, it can be seen that all the expressed protein spots on the three gels are with the same range of molecular weight (MW) and PI in terms of 17-80 KDa and 4.00-8.40, respectively. However, the detected spot numbers on day 1 (Fig.1-A), day 2 (Fig.1-B), and day 3 (Fig.1-C) are 160, 195, and 176, respectively. From Fig.2, all the detected spots are independent, indicating that the proteins are fully separated.

Fig. 1 Profile of the 2-DE analysis of the eggs of the high royal jelly producing worker bees (Apis melliferu L.) on different days. A, B, and C are the protein profiles corresponding to day 1, day 2, and day 3, respectively. Each sample of 77.4 pg was subjected to 2-DE and stained with CBB R-250.

Fig. 2 Three dimensional (3-D) view of the protein pattern and separation based on the same region in the 2-DE profile (Fig.1) on different days of the high royal jelly producing worker bees (Apis mellifera L.). A,B, and C are the 3-D patterns of day 1, day 2, and day 3, respectively.

02007,CAAS. Ail rights reserved.Publishedby Elsevier Ltd.

ZHANG Lan er al.

1142

Comparison of the protein pattern of the eggs of the high royal jelly producingworker bees (Apis mellifera L.) on different days By comparing the 2-DE profiles of the eggs on different days, 44 spots can be clearly matched to every profile, MW of these spots distributed in a wide spectrum from 19 KDa to 77 KDa, whereas, their PI concentrated within a relatively narrow scope from 5.27 to 8.06 (Table 1). Among these 44 spots, it can be observed

that the expressional volume of 16 spots is in an uptrend following the egg developmental process, but the volume of six spots is in a downtrend along with the egg developmentalprocess, whereas, the volume of 17 spots reaches a peak on day 2, and five spots are with a minimal expressional volume on day 2 (Table 1). Eighty-nine spots were specified on day 1, and their MW and PI ranged between 22-85 KDa and 5.22-8.40, respectively, and the expressional volume ranged from 330 to 46900 (Table 2). Next, 77 spots were specific to

Table 1 MW, PI, and expressional volume of the coexistent proteins of the eggs of the high royal jelly producing worker bees (Apis mellifera L.) on different days Spot No. (SSP) 0304 1001 1504 1505 2002 2208 2305 2706 3005 6104 7003 7106 7205 7605 8207 8511 1102 2304 2404 4704 5605 7306 1301 2505 2604 2806 3703 3704 4001 4002 5405 6306 6308 7105 7307 7506 7509 8604 8605 5809 6605 7006 7308 8306

Expressional volume

MW (KDa)

PI

41.9 25.0 50.6 49.3 22.5 32.6 33.5 68 19.4 29.04 20.8 29.6 31.6 57.4 30.3 52.2 28.4 41.7 44.7 66.1 58.2 34.2 33.8 49.7 58.1 77.3 67.1 66.3 24.5 22.1 46.2 40.4 34.6 29.5 34 50.2 48.3 54.3 55.5 79.3 58.45 26.6 39.7 37.1

5.27 5.44 5.42 5.62 5.69 5.86 5.63 5.7 6.19 6.91 7.07 7.11 7.11 7.16 7.99 7.59 5.52 5.87 5.82 6.39 6.6 7.18 5.47 5.85 5.85 5.78 6.02 6.26 6.38 6.44 6.52 6.86 6.83 7.4 7 7.01 7.55 8.06 7.77 6.56 6.77 7.36 7.12 7.64

Day 1

Day 2

Day 3

3 291 1733.7 1431.1 2266.1 10808.1 1481.5 2838.1 3 495.9 1121.7 2237.8 5631.1 10090.3 1161.3 11 498.9 728.5 1648.8 7787.7 14591.2 18 286.3 43 804.3 30 090 68 345.4 2523.1 34 194.7 21 265.9 3 336.2 14489.7 18947.6 21 578.8 7 236.3 5 960 4 099 5 094.2 8602.2 14710.5 22661.6 4707.7 1462.6 1131.7 39 378.7 27 832.8 17605.6 2525.4 4811.7

25 832 3 650.7 8 992.1 12783.7 26 741.9 6 432 4 372.6 9989.6 4723.7 17287.3 6506.8 17 282.4 1742.9 19652.2 3813.8 2 483.4 4R13.1 13060.9 17 182.7 19656.5 23 468.8 66 994.4 4 837.8 40 068.1 71 430.2 10 869.8 15 607.7 23 446.8 76553.8 18711.9 8 783.9 2062.5 21 800.9 21 394.6 32626.5 33 963 14516.4 8268.2 5 747.3 4 584.4 4 309.6 8 498 2216.3 3361.1

27 734.5 7454.7 23 427.7 35 974 27691.3 12 691.5 12 803 11 603.9 7331.1 17 639.4 7 576.3 35 586.8 4092.6 35 569.2 5662.2 8851.2 3 817.4 6573 8206.2 6967.1 12415.8 58078.2 1312.6 11 739.4 5 984.4 8569.6 3 367.2 169 45 123 16624.7 6 191.6 453.8 6 848.9 4592.5 18 403.1 30286.7 5 124.3 948.2 4 168.9 9 680 20301.8 9356.8 2524.6 5 668.1

02007,CAAS. All rightsresewed. Publishedby Elsevier Ltd.

Profile Analysis of the Proteome of the Egg of the High Royal Jelly Producing Bees (Apis mellijera L.)

the profile on day 2, and their MW scattered from 18 to 80 KDa, whereas, their PI was in the scope of 4.75-8.30, and the expressional volume was 140-39000 (Table 3). In addition, 80 spots existed on the profile on day 3 specifically, and their MW and PI were 18-80 KDa and 4.70-8.15, respectively, and the expressional volume ranged from 360 to 24 000 (Table 4). Twenty-four spots with MW 17-70 KDa and PI 5. 09-8.04 were both expressed on day 1 and 2, but silenced on day 3, whereas, 30-50 KDa accounted for

1143

70%. Among them, the expressional volume of the 15 spots with MW 17-70 KDa and PI 5.09-7.98 was in an uptrend on the second day compared with that on day 1. At the same time, the expressional volume of the nine spots with MW 30-55 KDa and PI 5.72-8.04 was in a downtrend on day 2 as compared with that of day 1 (Table 5). Forty-nine spots with molecular weights 18-71 KDa and PI 4.8-7.67 were silenced on day 1, at the same time both expressed on day 2 and 3. Among these

Table 2 MW, PI, and expressional volume of the specific proteins of the eggs of the high royal jelly producing worker bees (Apis rnelliferu L.) on day 1 Spot No. (SSP) 0208 1006 1405 1610 161 1 2007 2008 2109 2210 2506 2507 2508 2708 2709 2808 2809 2810 281 1 3006 3206 3408 3506 3607 3608 3609 3705 3806 3807 3808 3809 4003 4309 4310 431 1 4405 481 1 5003 5207 5312 5406 5507 5607 5708 581 1 5902

MW (KDa) 33.8 26.1 47.4 55.8 55.8 26.6 22.5 28.8 33.1 49.2 48.6 51.1 70.0 64.9 79.4 71.8 71.4 74.6 22.5 32.9 45.6 50.1 57.9 54.7 57.6 67.0 77.5 77.9 73.2 73.6 26.9 34.4 39.3 41.8 43.1 70.5 24.7 32.7 42.1 43.7 49.2 54.1 67.0 80.4 84.6

PI 5.22 5.50 5.56 5.41 5.66 6.02 5.91 5.84 5.75 5.67 5.91 5.83 5.79 6.00 5.85 5.72 5.97 5.96 6.11 6.16 6.03 6.13 6.21 6.01 6.01 6.14 6.02 6.14 6.01 6.14 6.41 6.38 6.37 6.33 6.38 6.33 6.49 6.49 6.49 6.63 6.52 6.52 6.58 6.43 6.53

Expressional volume 1249.4 6008.4 3312.6 1824.0 3955.1 7 848.4 8088.1 2171.5 8 263.0 4 632.6 28668.9 2 146.2 7 360.7 3216.5 4353.0 1680.1 22383.3 3 333.4 1366.6 6058.3 8 506.5 9 135.8 46906.8 5 674.7 2005.5 33 299.9 3 198.8 2036.7 921.8 7069.4 25818.8 2793.8 2 390.3 4425.7 23 569.4 3 854.8 16498.9 337.2 13 380.6 26478.9 2771.7 21341.8 12057.4 38 359.2 4558.1

kSpot No. (SSP)

6006 6007 6106 6205 6206 6207 6309 6409 6410 641 1 6506 6609 6610 6611 7010 701 1 7206 7405 7406 7510 7608 8112 8113 8208 8209 8308 8309 8310 8311 8404 8406 8512 8513 8514 8606 8801 8802 8803 9004 9302 9303 9304 9509

1 I

MW (KDa) 85.1 24.5 22.3 28.8 30.9 32.5 32.2 34.4 43.4 44.0 46.8 49.8 57.2 55.1 59.1 24.5 26.0 31.4 44.5 43.7 51.7 59.1 29.3 29.3 31.9 33.7 37.6 37.3 42.1 42.6 45.1 42.9 53.0 48.6 50.8 55.8 73.6 74.1 77.0 23.4 36.2 35.1 36.2 50.8

PI 6.43 6.84 6.83 6.68 6.66 6.87 6.67 6.68 6.83 6.12 6.68 6.76 6.92 6.71 6.72 7.02 7.37 7.23 7.30 7.00 7.29 7.33 8.00 7.89 7.52 7.70 7.55 7.44 7.65 7.55 7.55 7.72 7.72 7.72 7.50 7.67 7.69 7.55 7.56 8.37 8.39 8.32 8.03 8.40

Expressional volume 7405.9 15096.1 670.1 1 861.2 2 176.2 2 366.1 701.3 9077.1 5 340.0 15 596.0 737.3 12035.8 28 624.9 5 540.4 1145.8 7938.1 831.6 3 381.2 2523.1 13 799.0 18 964.0 9366.1 638.0 6281.2 6809.3 4418.3 4912.9 1172.6 1006.3 1088.1 1 843.4 5 760.5 4918.5 2 287.0 991.7 4023.7 8 343.7 383.0 798.5 1468.2 3 750.3 1164.7 1056.8 12 125.3

ZHANG Lan et al.

1144

Table 3 MW, PI, and expressional volume of the specific proteins of the eggs of the high royal jelly producing worker bees (Apis mellifera L.) on day 2 ~

Spot No. (SSP)

000 1 0102 0206 0606 1002 1004 1005 1302 I606 1608 1707 1708 1806 1807 2005 2206 2207 2306 2605 2707 2807 3406 3407 3504 3505 4104 4208 4505 4506 4606 4607 4608 4705 4706 4806 4807 4808 4809

MW (KDa) 17.9 29.3 31.1 58.4 21.7 26.0 26.5 35.6 60.3 54.1 69.4 60.8 79.1 78.9 26.8 33.3 32.7 41.0 53.0 69.8 71.7 43.8 43.1 52.7 52.5 28.9 32.3 51.9 52.1 60.2 54.2 54.2 70.5 60.4 77.4 77.9 78.5 80.8

PI 5.34 4.75 5.30 5.27 5.57 5.60 5.46 5.48 5.50 5.39 5.57 5.40 5.60 5.40 5.82 5.74 5.66 5.71 5.85 5.87 5.66 6.03 6.17 5.99 6.10 6.33 6.38 6.35 6.45 6.36 6.30 6.38 6.29 6.42 6.29 6.37 6.42 6.46

Expressional volume

3 567.5 908.8 141.0 2 357.5 67.5 499.4 1396.5 695.9 9 190.3 5 884.1 10 256.5 4 998.7 686.9 2 250.7 2 101.8 5 960.0 4713.1 4 193.8 5 709.4 39066.3 9578.8 18 188.0 30 970.5 1038.1 28 360.9 1306.0 8260.1 2320.3 2211.4 20 620.1 20 023.1 21 431.3 17 161.2 22951.0 3 488.7 4663.1 4 020.3 20 188.8

spots, the expressional volume of the 28 spots with molecular weight 18-71 KDa and PI 4.8-7.64 were upexpressed on day 3 as compared with those on day 2, and the expressional volume of the 21 spots with molecular weight 19-71 KDa and PI 5.28-7.67 were downexpressed on day 3 compared with those on day 2 (Table 6). In addition, there were three spots that were silenced on day 2, whereas, expressed both on day 1 and 3. Among the three spots, the expressional volume of only one spot (MW 45.4 KDa, PI 7.62) increased on day 3 compared to that on day 1, and the volume of two spots (MW 45.4 KDa, PI 6.17; MW 53.0 KDa, PI 6.38) decreased on day 3 as compared to that on day 1 (Table 7).

Spot No. (SSP)

4810 5001 5102 5308 5309 5310 5311 5504 5506 5604 5606 5706 5810 6003 6005 6105 6203 6407 6505 6606 6608 7004 7009 7508 7606 7607 8001 8110 8111 8510 9001 9002 9003 9102 9207 9208 9209 9508

Mw( m a ) 70.8 25.9 28.7 39.4 35.8 35.3 33.6 49.4 50.0 58.4 55.7 68.7 80.9 17.5 19.2 30.1 32.0 47.1 47.8 56.1 57.9 21.2 24.4 50.5 57.7 53.8 20.7 28.7 27.7 50.5 20.9 20.9 21.0 29.4 30.7 32.2 30.4 51.0

~

PI 6.39 6.63 6.60 6.52 6.50 6.62 6.56 6.47 6.64 6.51 6.61 6.55 6.61 6.92 6.89 6.75 6.76 6.95 6.76 6.74 6.90 7.10 7.39 7.37 7.35 7.18 8.03 7.88 7.84 7.72 8.08 8.24 8.30 8.08 8.24 8.26 8.12 8.26

~~

Expressional volume 15 924.6

1431.9 2061.5 3 563.1 3 137.4 1384.6 6846.0 8934.8 3 933.0 234.3 18 880.4 20 978.0 5 009.0 4011.8 1207.3 3 552.8 5314.5 2 320.5 11 566.4 397.4 18 822.3 1831.5 863.4 881.9 3 427.6 1 189.2 704.5 4426.3 5 110.6 3 868.2 2821.9 5 534.0 338.4 7 557.1 14 089.5 7 878.7 7686.5 11 915.6

DISCUSSION The 2-DE-based proteome analysis has been successfully used to detect and characterize marker proteins of a cell or tissue (Zivy and de Vienne 2000). To date, only five articles are available, obtained by employing the proteomic approach, to detect and identify the proteins of royal jelly proteome (Sano et al. 2004; Santos et a l . 2005; Scarselli et al. ZOOS), the spermathecal gland secretion, as well as spermathecal fluid in the queen bees (Klenk et al. 2004), and the proteins of the honeybee venom (Peiren et al. 2005). The present study is a pioneer one that uses the proteomic assay to compare the proteome complement in the developmental process of the worker bees' egg

02007, CAAS. All rights re8efved.Publishedby Elsevier Ltd.

Profile Analysis of the Proteome of the Egg of the High Royal Jelly Producing Bees (Apis rnelliferu L.)

1145

Table 4 MW, PI, and expressional volume of the specific proteins of the eggs of the high royal jelly producing worker bees (Apis rnelliferu L.) on dav 3 Spot No. (SSP) 0002 0003 0209 0210 0306 0504 0607 0608 1007 1008 1303 1406 1506 1507 1612 1613 2009 2010 2110 221 1 2308 2405 2606 2710 2812 3007 3507 3610 3706 3707 3810 381 1 4004 4107 4609 5004 5005 5208 5313 1608

MW ( m a )

PI

Expressional volume

18.2 22.7 31.8 33.6 42.5 50.3 59.6 54.2 23.2 22.6 35.6 44.7 53.9 48.8 59.2 56.3 17.8 21.3 29.7 33.7 35.6 44.1 57.8 69.4 80.2 21.0 52.6 59.2 60.0 68.1 80.7 70.2 20.6 28.9 56.7 20.1 24.5 30.5 37.0

5.32 5.11 5.07 5.12 5.08 4.70 4.84 5.13 5.37 5.50 5.66 5.63 5.41 5.50 5.51 5.55 5.84 5.80 5.74 5.85 5.84 5.71 5.70 5.91 5.87 6.13 6.13 6.05 6.16 6.16 6.17 6.02 6.36 6.36 6.38 6.60 6.61 6.53 6.50 6.50

1247.8 1183.5 3080.3 2756.5 2634.2 8945.1 7 842.0 4044.0 678.0 7 623.4 7 189.2 7613.0 6930.9 5613.8 4556.4 3 152.0 14622.0 757.1 3466.6 9754.8 10739.1 4477.1 19979.8 20486.6 5453.1 3 483.9 8 830.5 10001.1 23564.1 18477.0 9700.3 5 655.2 2753.2 2465.8 2 146.9 1663.6 3 342.9 3 299.3 7514.9 11 045.7

59.2

1 Spot No. (SSP) 5709 5710 571 1 5812 6008 6107 6310 6412 6612 6613 6614 6705 6804 6805 6806 7012 7013 7107 7207 7407 7609 7705 7804 7805 7 807 8002 8003 8004 8005 8006 8114 8115 8210 8312 8407 8515 8516 8607 9103 119510

MW (KDa)

PI

63.5 66.4 69.8 72.9 22.1 29.8 34.0 43.5 56.3 56.0 58.5 68.9 80.7 73.4 71.1 21.6 26.6 30.3 33.9 43.8 56.0 67.2 79.2 76.7 72.5 16.8 17.4 19.2 23.9 24.6 28.9 30.0 32.2 34.0 45.8 51.2 52.9 56.0 29.9 51.6

6.58 6.58 6.55 6.53 6.68 6.81 6.63 6.66 6.85 6.72 6.70 6.75 6.68 6.71 6.86 7.37 7.05 7.37 7.33 7.02 7.00 7.21 7.33 7.15 7.01 7.52 7.50 7.54 7.67 7.44 7.88 7.74 7.91 7.51 8.02 7.87 7.67 7.5 1 8.14 8.13

Expressional volume 6711.0 6 796.1 3 689.7 945.0 1516.1 5 006.5 3896.1 8 590.1 19 112.1 14619.0 13 866.5 2 359.2 1261.5 1415.9 14721.7 3 599.8 3 984.1 10701.0 22 872.4 3 987.5 6 830.6 3901.2 4 754.2 1387.7 2668.2 1956.8 267.5 1698.7 3 690.7 7 806.8 4305.3 7 730.3 2277.5 2770.7 2 379.1 4485.8 364.6 821.9 6 130.1 4742.5

Table 5 MW, PI, and the expressional trend of the eggs of the high royal jelly producing worker bees (Apis rnelliferu L.) expressed on day 1 and day 2, but silenced on day 3 Spot No. (SSP)

0605 1706 2004 2307 3004 3307 3805 4404 5705 6607 7204 7703 8206 8307 8509 2106 3606 5206 6001 6204 8205 2504 6504 7309

MW ( m a )

PI

55.9 67.6 17.5 33.9 26.8 39.3 70 8 43.1 70.5 56.0 33.5 63.5 30.3 38.8 51.8 29.5 58.1 30.7 26.6 31.1 33.6 52.5 50.5 34.6

5.09 5.41 5.78 5.83 6.19 6.18 6.24 6.27 6.47 6.87 7.5 I 7.31 7.77 7.98 7.84 5.85 6.10 6.52 6.85 6.96 8.04 5.72 6.91 7.38

Day 1 1173.1 3020.7 3 739.1 1681.5 7 877.3 627.3 3 607.4 4000.0 10346.0 738.4 1299.2 919.8 10775.7 1918.7 7 932.9 7 796.5 58270.6 4274.5 6300.3 3617.2 7 899.2 2 809.0 3 046.1 20039.4

Expressional volume Day 2 2374.9 15 095.8 7 590.0 5 769.1 9377.2 1134.5 21 319.8 30 685.9 17 649.6 5483.1 3 222.2 1506.9 21 064.6 3 809.7 9 194.5 2588.8 15715.4 2487.6 946.0 1301.7 6923.7 2516.1 2861.2 19789.8

Day 3

(92007.CAAS. All rights resewed. Publishedby Elsevier Ltd.

ZHANG Lan et ul.

1146

Table 6 MW, PI, and the expressional trend of the eggs of the high royal jelly producing worker bees (Apis melliferu L.) expressed on day 2 and day 3, but silenced on day 1 Expressional volume Spot No. (SSP) 0101 0204 0205 0305 1204 1607 1609 2001 2006 2101 2107 2108 3003 3101 3104 3205 3308 3605 4105 4207 4306 5205 5505 6004 6408 7001 7008 8109 0207 1003 2003 2209 3001 3002 3804 4209 4307 4308 4507 4605 5707 6002 6307 6503 7007 7404 7507 7704 8702

MW (KDa)

PI

27.1 32.1 30.6 34.5 32.9 56.2 57.0 19.9 24.6 26.9 28.1 28.1 24.6 27.0 29.0 30.6 34.0 55.5 27.5 33.6 41.1 32.0 51.7 18.2 43.7 20.1 24.7 29.7 30.5 21.9 18.8 30.6 20.4 23.5 71.2 31.2 41.4 37.1 48.8 57.6 68.5 20.9 38.2 50.5 19.1 45.9 49.9 67.6 67.8

4.80 5.14 5.13 4.79 5.37 5.61 5.40 5.67 5.83 5.64 5.83 5.68 6.08 6.00 6.1 1 6.03 6.18 6.17 6.36 6.37 6.21 6.59 6.56 6.71 6.76 7.04 7.11 7.64 5.28 5.59 5.77 5.87 6.00 6.02 6.16 6.27 6.40 6.33 6.34 6.29 6.65 6.89 6.71 6.82 7.23 7.30 7.26 7.41 7.67

Day 1

Day 2 2 879.2 1741.9 1236.1 2423.5 1804.9 3691.8 3 390.9 3482.0 3 763.2 1977.4 3 537.7 1137.0 7 809.0 33 823.0 1361.1 1006.6 5092.8 5 139.3 3 994.9 6319.6 8793.8 3 267.4 7 107.0 563.7 26528.7 2550.2 1120.9 10032.1 2416.6 1519.4 2 397.4 12305.8 1778.3 10767.6 10780.2 8 131.0 10308.9 16388.1 2 184.2 5981.6 16047.8 1549.9 11531.2 13 998.5 3 246.2 6 266.6 9015.5 3 843.3 5568.3

Day 3 5 593.3 2112.8 5813.3 2 872.2 2660.6 31 258.6 19987.9 7 182.9 5 377.6 6 108.9 7987.2 1937.6 10498.5 39523.9 5 794.2 2 175.0 24 758.2 12 156.7 11 682.3 12 189.9 10558.2 9810.6 8 762.0 1079.8 33 206.0 10334.3 2 760.0 13 252.3 1647.3 1019.4 1616.0 2 166.7 1380.9 10296.1 1580.6 2 109.7 3 184.6 3 548.7 1287.3 4034.8 3 167.0 87 1.4 4499.4 10902.1 306.8 2989.2 3 737.6 3 774.6 1620.3

Table 7 MW, PI, and the expressional trend of the eggs of the high royal jelly producing worker bees (Apis melliferu L.) expressed on day 1 and day 3, but silenced on day 2 Spot No. (SSP) 8405 3409 4508

(ma) 45.4 45.4 53.0

PI 7.62 6.17 6.38

ExDressional volume Dav 1 3 706.0 11551.8 9969.6

Dav 2

Dav 3 10789.0 5 327.6 2588.1

02007,CAAS. All Chts reserved. Publishedby ElsevierLtd.

Profile Analysis of the Proteome of the Egg of the High Royal Jelly Producing Bees (Apis melliferu L.)

of the high royal jelly producing bees (Apis mellifera L.). Royal jelly production is a colony performing trait of the worker bees (Graham 1992). Previous studies showed that royal jelly production is dominated by genetic components (Li et al. 2003a, b; Li and Wang 2005). Therefore, this genetic process must begin from the egg development of the worker bees. The egg’s cleavage begins from when it is laid to the cell. The egg divides to initiate a succession of nuclear cleavage divisions and forms cellular layers. The organs of the bees are derived from these layers, and help to develop the embryo. By the time of day 3 after the egg is laid, embryologicaldevelopment is complete and the egg now contains a larva. But, the change of modality of the worker bees is insignificant along with the process of egg development (Harry and Robert 1996). On the basis of the detected protein spots along with the egg developmental process, it can be seen that egg development is a sequential and complex gene controlled process, which involves many proteins, although the modal change is insignificant. It can be seen that the eggs of day 2 express most actively, 160, 195, and 176 proteins expressed on day 1, day 2, and day 3, respectively, indicating that the genes in the egg have been activated in an environment at 34°C when the egg is laid to the cell, and activated genes are less on day 1 and more on day 2 and 3, and prepare to hatch, along with the three-day development of the high royal jelly producing bees (Apis melliferu L.). The 44 protein spots are constantly detected along the process of the development of the eggs indicate that these proteins are conservative, and are indispensable for this program. But these coexisting proteins are not all the same; their expressional volume has some changes. Thirty-six percent of the coexisting proteins are in the uptrend along with the egg development, and 39% of them are of the largest expressed volume on day 2. It can be seen that these coexisting proteins regulate the development of the egg by way of altering the expressional volume. It also indicates that egg development is a sequential and complex gene controlled dynamic process. Nowadays, the reports concerning the basic research on the egg development of the bees are not enough, and the relationship between the change of modality

1147

and the structure of the egg, with the expression and the regulation of the proteins is still not clear. The present experiment just lays a preliminary foundation in the proteome of the egg development of the high royal jelly producing bees (Apis melliferu La). If all the proteins can be sequenced by mass spectrometry (MS) and identified by database querying, it will benefit to further understand the mechanism of egg development of the high royal jelly producing bees (Apis mellifera L.). In addition, if the differential proteome can be compared between the high royal jelly producing bees (Apis mellifera L.) and the unselected ones (Apis melliferu L.), the functional gene will be found. All of these will be conducive to elucidate the molecular mechanism concerning the high royal jelly production and will lay a foundation for molecular breeding.

Acknowledgements This work was supported by National Key Technologies R & D Program during the 11th Five-Year Plan Period of Ministry of Science and Technology, China (2006BAD06B04,2006BAD12B08-06).

References Bradford M M. 1976. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein dye binding. Analytical Biochemistry, 72,248-254. Chen S B, Han S M. 1992. Summary of the production capacity of the high royal jelly producing bee. Apiculture ofChina, 43, 2-5. (in Chinese) Chen S L, Li J K, Zhong B X, Su S K. 2005. Microsatellite analysis of royal jelly producing traits of Italian honeybee (Apis mellifera L.). Acta Genetica Sinica, 32, 1037-1044. Chen S L, Lin X Z. 1995. Experiment on the higher production royal jelly bee. Scientia Agricultura Scenica, 28, 89-93. (in Chinese) Dai Y, Xu X, Wang C F, Jiang Y. 2003. Study on transforming from RAPD molecular marker-W316bp of high royal jelly products into a new SCARSmolecular marker. Apiculture of China, 54,7-8. (in Chinese) Fleig R, Walldorf U, Gehring W J, Sander K. 1992. Development of the deformed protein pattern in the embryo of the honeybee Apis mellifera L. (Hymenoptera). Development Genes and Evolution, 201, 235-242. Graham M J. 1992. The Hive and the Honeybee. Dadant & Sons, Inc., Illinois. pp. 73-101. Harry H L, Robert E P. 1996. Queen Rearing and Bee Breeding.

02007, CAAS.All rights reserved. Published by ElsevierLM.

1148

Wicwas Press, Cheshire. p. 26. Jiang Y, Zhang Y J, Jiang J X, Xue Y B, Dai Y, Xu Y. 2002. Study on identification of special marker in DNA molecule of high royal jelly bee. Journal of Bee, 8, 3-4. (in Chinese) Jin S H, Zhang D L, Xu Y, Wu J C, Jiang Y. 2003. Study on identifying high royal jelly products bee (Apis mellifera L.) using molecular marker-W316bp. I .Apiculture ofChina, 54, 7-8. (in Chinese) Jin S H, Zhang D L, Xu Y, Wu J C, Jiang Y. 2004. Study on identifying high royal jelly products bee (Apis mellifera L.) using molecular marker-W316bp. I1 .Apiculture of China, 55, 4-5. (in Chinese) Klenk M, Koeniger G, Koenige N, Fasold H. 2004. Proteins in spermathecal gland secretion and spermathecal fluid and the properties of a 29 KDa protein in queens of Apis mellifera. Apidologie, 35, 371-381. Li J K, Chen S L, Zhong B X, Su S K. 2003a. Genetic analysis for developmental behavior of reproductive ability and hypopheryngeal gland in western honeybees (Apis rnellifera L.). Chinese Journal ofAnimal Science, 39,9-11. (in Chinese) Li J K, Chen S L, Zhong B X, Su S K. 2003b. Genetic analysis for developmental behavior of honeybee colony’s royal jelly production traits in western honeybees. Acta Genetica Sinica, 30,547-554. (in Chinese) Li J K, Wang A P. 2005. Comprehensive technology for maximizing royal jelly production. American Bee Journal, 145,661-664. Liu Y H, Chen S L, Zhong B. 2001. The combining ability and heterosis analysis of royal jelly yield and quality properties in western honeybees. Acta Genetica Sinica, 28,926-932. (in Chinese) Peiren N, Vanrobaeys F, de Graaf D C, Devreese B, van Beeumen J, Jacobs F J. 2005. The protein composition of honeybee venom reconsidered by a proteomic approach. Biochimica et Biophysica Acta, 1752, 1-5. Ronglin Y, Omholt S W. 1999. Early developmental processes in the fertilised honeybee (Apis mellifera) oocyte. Journal of Insect Physiology, 45,763-767. Sano 0, Kunikata T, Kohno K, Iwaki K, Ikeda M, Kurimoto M. 2004. Characterization of royal jelly proteins in both Africanized and European honeybees (Apis mellifera) by twodimensional gel electrophoresis. Agricultural and Food Chemistry, 52, 15-20. Santos K S, dos Santos L D, Mendes M A, de Souza B M, Malaspina 0, Palma M S. 2005. Profiling the proteome complement of the secretion from hypopharyngeal gland of Africanized nurse-honeybees (Apis mellifera L.).Insect Biochemistry and Molecular Biology, 35,85-91. Scarselli R, Donadio E, Giuffrida M G, Fortunato D, Conti A,

ZHANG Lan et al.

Balestreri E, Felicioli R, Pinzauti M, Sabatini A G, Felicioli A. 2005. Towards royal jelly proteome. Proteomics, 5,769776. Shen J K, Xiao T Y. 1993. Experimental report on the production performance of the high royal jelly producing bee. Apiculture of China, 44,4-6. (in Chinese) Su S K, Chen S L. 2003. Research on morphological genetic marker of honeybee (Apis mellifera L.) in royal jelly production performance. Hereditas, 25,677-680. (in Chinese) Su S K. 2000. Currency of genetic marker of honeybee (Apis mellifera L.) in royal jelly production performance. Apiculture of China, 51, 8-9. (in Chinese) Tamar K G, Soroker V, Kamer J, Schulz C M, Francke W, Hefetz A. 2003. Ultrastructural and chemical characterization of egg surface of honeybee worker and queen-laid eggs. Chemoecology, 13,129- 134. Wang W P, Jiang Y, Zhang Y J, Wang C F, Huang C. 2002. Selection and checking of the gene marker related to the high royal jelly quantity trait of Apis mellifera Lindauer. Chinese Journal of Biochemistry and Molecular Biology, 18, 161164. (in Chinese) Xu Z Y, Shao F Q, Li S X, Chen X Q. 2000. Study on utilizing of the cross-breed in higher honey and royal jelly production western honeybees (Apis mellifera L.). I .Apiculture of China, 51, 10-12, 18. (in Chinese) Xu Z Y, Shao F Q, Zhang R W, Li J S, Li H Q, Zhao W Y, Chen X Q. 2001. Study on utilizing of the cross-breed in higher honey and royal jelly production western honeybees (Apis mellifera L.). 11. Apiculture of China, 52,9-11. (in Chinese) Zhang Y J, Jiang Y, Wang W P, Ge F C, Wang C F, Zhang D L. 2001a. Study on identifying the special marker of DNA in Apis mellifera of high or low royal jelly products with probe dig-W316bp. Apiculture of China, 52,6-8. (in Chinese) Zhang Y J, Jiang Y, Wang W P, Ge F C, Wang C F, Zhang D L. 2001b. Study on a special marker-W316bp in Apis mellifera Lindauer of high royal jelly products. Apiculture of China, 52,7-9. (in Chinese) Zhang Y J, Jiang Y, Wang W P, Ge F C, Wang C F, Zhang D L. 2001c. Study on preparation of DNA probes (W316bp) marked with digoxigenin. Apiculture of China, 52, 10-11,23. (in Chinese) Zhong B X, Li J K, Lin J R, Liang J S, Su S K, Xu H S, Yan H Y, Zhang P B, Fujii H. 2005. Possible effect of 30 K proteins in embryonic development of silkworm Bombyx mori. Acta Biochimica et Biophysica Sinica, 37,355-361. Zivy M, de Vienne D. 2000. Proteomics: a link between genomics, genetics and physiology. Plant Molecular Biology, 44,575580. (Edited by WANG Lu-han)

02007,CAAS. All tights reserved.Publishedby Elsevierltd.